The field of the present invention is that of biological analysis, in particular hematological analysis. The invention relates to the general field of devices and methods for electro-optic measurements in a fluid present in a measuring tank. Such devices and methods are intended in particular for classifying and counting microscopic objects in a fluid, for example a biological fluid.
To be more precise, the invention relates to devices that use analysis methods based on using electrical and optical measurements to count and distinguish cells present in a sample to be analyzed. In the context of the present invention, this is preferably a blood sample.
With hematopoietic cells, the person skilled in the art knows that volumetric or diffractometric morphological analysis of the cell, including the phenomena of extinction or absorption, enables discrimination of the main cell lines includes erythrocytes (red cells), thrombocytes (platelets), and leucocytes (white cells). The white cell population is itself divided into a plurality of categories such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.
The maturity of these cells can to some extent be determined by determining simultaneously their volume and their apparent absorption of white light, as described in U.S. Pat. No. 5,138,181 filed by the Applicant. A device using quasi-monochromatic light is described in the patent WO 2006/053960.
This assessment of cellular maturity is very important because it enables early diagnosis. Generally speaking, most cells present in the circulating blood are mature cells.
For each of the above-mentioned cell types, the various levels of maturity are known. Thus red cells, also called erythrocytes, are first produced in the form of proerythroblasts, then basophilic erythroblasts, then polychromatophilic erythroblasts which evolve into acidophilic erythroblasts, then into reticulocytes. It is these reticulocytes that finally differentiate into erythrocytes once they have entered the circulating blood.
White cells, also called leucocytes, are also first produced in the bone marrow in the preliminary myeloblast form. These myeloblasts thereafter yield the progranulocyte that is then transformed into basophilic, eosinophilic or neutrophilic granulocyte, at first non-segmented and then with a nucleus that segments increasingly with age.
The same myeloblasts are also the source of the monocyte line that yields the monoblasts, promonocytes and then monocytes that enter the peripheral circulation.
The stem cells from which the myeloblasts originate also give rise to the lymphocyte line through a differentiation in lymphoid stem cell form, part of which line continues to mature in the thymus and the ganglions (line T) and the remainder remains in the bone marrow in order to yield the B lymphocyte line. The same B lymphocytes that, once activated in the form of plasmocytes, produce the antibodies to combat pathogenic antigens.
Blood platelets, also called thrombocytes, are derived from the megacaryoblasts, themselves stemming from the myeloid progenitor from which originate the myeloblasts which, on reaching the final stage of their maturation (thrombocytogen megacaryocytes) produce platelets by splitting their cytoplasm. The recent platelets (reticulated platelets) contain RNA that is the residue of the original cell.
The diagnosis of some pathologies requires the increasingly refined counting of hematopoietic cells. In particular, it becomes necessary to be able to show up new populations such as reticulocytes and erythroblasts that are the immature versions of erythrocytes. Similarly, showing up immature cells, precursors of the leucocytes, called immature lymphocytes, monocytes or granulocytes is of great importance. Likewise, classifying and counting the activated lymphocytes or reticulated platelets would make it possible to obtain a meaningful improvement in the diagnosis of patients.
Since the mean lifetime of a red cell is 120 days, the normal regeneration rate must therefore be 0.83%. The normal mean percentage generally accepted is in the range 0.5% to 1.5%, these values being higher (from 2% to 6%) in the neonate of less than 3 weeks. Observing and counting the reticulocytes is thus an indicator of erythropoietic activity and thus a parameter that is especially useful, in particular in monitoring medullary regrowth after chemotherapy, in follow-up treatment by recombinant erythropoietin (rHuEpo), in the anemia exploratory balance or in searching for a hemolysis or compensated hemorrhagia.
A few examples of pathologies in which differentiating and counting cells in this way are useful are given below.
The clinical benefit of counting erythroblasts may prove important for detecting certain forms of anemia, for example. The common characteristic of hemolytic anemia is the excess destruction of adult erythrocytes that may result from extracorpuscular factors or intrinsic anomalies of the structure or function of the erythrocytes.
Erythroleukemia is a variety of acute myeloblastic leukemia that is characterized by malignant proliferation of cells of the red line and precursors of the granular line. From the cytological point of view, peripheral hyperleucocytosis is noted at the initial stage with circulating blasts present (59%). The myelemia corresponds to the passage into the circulating blood of immature elements, the granular line or the erythrocyte line.
On the subject of differentiation, reticulocytes constitute one of the most interesting cell families and numerous methods exist and are under development in automated hematological analysis. These cell lines may be counted by marking nucleic acids by means of fluorescent coloring agents, for example asymmetrical cyanines, and in particular Thiazole Orange.
This molecule has unique physico-chemical characteristics for intra-cytoplasmic detection of nucleic acids (RNA or DNA). When free in solution, this molecule exhibits very little fluorescence induced by photo-excitation. The stereochemical configuration of Thiazole Orange is such that this molecule is interleaved between the bases of the nucleic acids. In this state the molecule fluoresces. Thus detecting and measuring the level of fluorescence makes it possible to quantify intra-cytoplasmic nucleic acids. The nature of the cells may be deduced from this quantification of intracytoplasmic nucleic acids combined with a second optical or electrical measurement, thus enabling them to be counted absolutely or relatively.
Existing electro-optic devices for measuring this fluorescence are relatively complex. They use considerable hardware resources, especially if high analysis rates are expected. In this situation, it is relatively standard practice to use a laser light source with a set of sensors for measuring diffraction, generally identified by the abbreviations FSC (forward scattering) and SSC (side scattering), or extinction, generally identified by the abbreviation ALL (axial light loss).
The same laser makes it possible to induce fluorescence of the marker or markers or the coloring agent or agents present on or in the cell at the moment of crossing the laser beam. In the standard manner, fluorescence light and diffraction light are separated on the basis of their spectral properties. To this end, multi-dielectric interference optical filters are generally used, i.e. filters obtained by the alternate deposition of two or more transparent materials having different refractive indices. To measure fluorescence photomultipliers or photodiodes are used that most of the time operate in avalanche mode. These systems are relatively complex from the optical and mechanical points of view.
US patent application 2006/0219873 discloses the use of an AGC (automatic gain control) avalanche photodiode. This device is used in a flow cytometer in which the gain of the photodiode is adjusted automatically as a function of the applied voltage.
Patent application EP 1 710 558 in the name of Sysmex discloses a plurality of sensors for recovering data from each light source. This device also effects only SSC measurements.
Patent application EP 0 533 333 discloses an on-line reader device in which the cells are not analyzed when flowing. Again, even though data may be obtained (absorption, fluorescence, reflectance data), the data is not obtained with a single sensor.
Patent application WO 2008/019448 discloses an epifluorescence device with no absorption reading.
Patent application EP 0 806 664 uses a plurality of sensors if it is necessary to obtain data from a plurality of different sources.
U.S. Pat. No. 4,745,285 discloses a device capable of counting particles marked with a plurality of fluorochromes. This device has a single-wavelength light source and a plurality of sensors capable of recovering the data of each fluorescence.
U.S. Pat. No. 5,408,307 discloses a device able to effect FSC, SSC and fluorescence measurements using plurality of different sensors.
U.S. Pat. No. 6,897,954 (Becton Dickinson and Co), describes the use of a plurality of fluorescences associated with a plurality of photosensors to count cells when flowing. The gain of each photosensor may be modulated in order to adjust it to the fluorescence detected.
None of the known devices integrates the electro-optical and opto-fluidic measuring systems necessary for differentiating and counting biological cells, in particular those in the circulating blood. The size and complexity of the existing devices make them costly and complex to operate.